1. Field of the Invention
The present invention relates to a dual laser excitation apparatus with a single laser source, and more particularly, concerns a flow cytometry apparatus for determining characteristics of cells or the like.
2. Related Information
There are many applications in which lasers are employed as light sources. One such application of lasers is in flow cytometry apparatuses. In a flow cytometry apparatus, cells or other particles are passed in a liquid flow stream so that one or more characteristics of the cells under investigation may be determined. Cellular analysis by reliance on optical investigation is a commonly used technique in flow cytometry apparatuses. Typically, an incident beam of light is directed at the stream of cells as they pass through the apparatus. The passing cells scatter the light as they pass through the light beam. Scattered light has served as a function of cell shape, index of refraction, opacity, roughness and the like. In addition, fluorescence emitted by labeled cells which have been excited as a result of passing through the excitation energy of the incident light beam is detectable for identification of specifically labeled cells. Lasers have been used as the source of the incident beam of illumination in flow cytometry apparatuses.
Multi-parameter analysis of cells is a tool which provides significantly more information about individual cells in order to understand the characteristics of such cells. One technique for improving multi-parameter analysis is the multiple fluorescence detection with respect to the cells under investigation. Although it has been known in the art that a single light source, such as a laser, may be used as an excitation source or two fluorescent labels on the cells, the increased need for multiple labels and the requirement that the absorption of the label match the wavelength of the laser has brought about development of systems which employ two lasers in order to excite two or more distinguishable fluorescent markers. Apparatuses utilizing two lasers for cellular analysis are described, for example, in U.S. Pat. Nos. 3,826,364 and 4,284,412.
Utilization of dual laser systems also permits the multi-parameter cell analysis and sorting with three or four different fluorochromes. One such dual laser system, known as the FACS 440 Cell Sorter manufactured and sold by Becton Dickinson Immunocytometry Systems, Mt. View, Calif., relies upon a common lens system for collecting fluorescence emission from all fluorochromes, after which individual colors are separated by means of appropriate combinations of dichroic filters, mirrors and beam splitters. This flow cytometry system is described in commonly assigned patent application Ser. No. 482,345, filed on Apr. 5, 1983. In order for such a flow cytometry system to obtain optimum performance in terms of signal to noise characteristics in each channel, and with minimum channel crosstalk, it is desirable that the two laser excitation sources have sufficient separation both spectrally and spatially. By spectral separation, it is meant that the second laser excitation wavelength should be outside the acceptance window of the emission filters for cells excited by the first laser.
In the flow cytometry system described in the aforementioned patent application, spatial separation is achieved by directing the two lasers so that the respective focal points on the cell stream are vertically displaced by about 200-250 microns. The pulses from the two lasers are thus time-separated. Spatial separation occurs by placing a high reflectance mirror in the optical emission path near a magnified image of the stream. This so-called split mirror is positioned so that emission from the first laser passes beyond the mirror's edge, while emission from the second laser is intercepted and reflected into an alternate path. Further spectral separation of the individual emission channels in the two spatially separated beams is accomplished by placing an appropriate dichroic in each light beam.
Adequate spectral separation of the excitation light sources has been obtained by using a visible laser, such as an argon laser, in single line mode as a primary laser, and a dye laser with a multi-line argon pump as the secondary laser. The primary laser may be adjusted to one of the visible argon lines between, for example, 457.9 nm and 514.5 nm, while the secondary laser uses a dye, such as for example, R6G which may be adjusted to output a line between 580 nm and 640 nm. For four color applications, the primary laser, which may, for example, be set at 488 nm, excites two fluorochromes, such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE) which emit fluorescence at 520 nm and 575 nm, respectively. On the other hand, the dye laser, which may, for example, be set 598 nm, excites two additional stains such as Texas red (TR) and allo-phycocyanin (A-PC) with corresponding emission peaks at 625 nm and 660 nm, respectively.
A scheme for obtaining dual line performance from a single dye laser system is described by Arndt-Jovin et al. in "A Dual Laser Flow Sorter Utilizing a CW Pumped Dye Laser," Journal of the Society for Analytical Cytology, vol. 1, no. 2, pp. 127-131 (1980). The performance of the scheme described by Arndt-Jovin et al. cannot be expected to be as good as a dual laser system for a number of reasons. First, short wavelength line isolation is done entirely with interference filters. Such filters with spectral band widths narrow enough to isolate a single argon line usually do not have a very high transmission peak, and it cannot be expected to withstand the required power densities for long periods of time. Second, changing the wavelength of the primary line is usually not a simple external adjustment, but rather requires changing several filters, and perhaps some realignment. Finally, the method of stabilizing the two laser lines simultaneously is usually inadequate.
There are other technical problems which are associated with the use of a single dye laser system for obtaining dual line performance. Current dye laser systems use the argon laser in a multi-line mode simply because this is the way to obtain the most available power for pumping the dye. Since such a system expects a single argon laser to satisfy the requirements of both the primary and secondary laser, the efficiency of the filtering system is critical with respect to the performance characteristics. Laser amplitude stability is also a problem. The best known method for stabilizing the output of lasers is what is known as light control in which a small amount of the final output beam is picked off and continuously monitored by a detector. This signal is used as feedback for controlling the input current for the laser. The individual output lines in a multi-line laser do not necessarily drift in the same way. Therefore, any system which spectrally splits up a single pump into two separate sources can accept feedback from only one of the final output lines as input current control. Thus, unless a special provision is made, at least one of the incident sources will be less stable than the normal dual laser system.
Previous schemes for using part of the pump for independent excitation, such as described by Arndt-Jovin et al., usually require changing several filters along with realignment for changing wavelength. Also, changing the wavelength of the short wavelength line has an impact on the dye laser performance. Moreover, narrow band interference filters usually do not withstand the high laser power densities for extended lengths of time without ultimately burning and becoming damaged, or useless.
Finally, present systems, which make no provision for independent focusing of the argon and dye optical paths onto the cell stream, result in a less than optimum focus of one of the two beams. The need for independent focus adjustment is occasioned by the substantially unequal output beam divergence of the respective pump and dye lasers. One dye laser system which is manufactured by Coherent Inc. of Palo Alto, Calif., has a pump output divergence of 0.00048 radians at 488 nm, and a dye output divergence of 0.0015 radians at 600 nm. Such a system cannot be simultaneously focused by a single lens, even a well corrected achromat.
It can be appreciated that improvements are needed in laser apparatuses wherein dual line performance results from a single dye laser system. The present invention is directed to such improvements and to the solution of the deficiencies and problems mentioned above.